Recombinant adeno-associated virus (rAAV) vectors have demonstrated transduction and long-term gene expression with little to no toxicity and inflammation in vivo. These unique characteristics of AAV have led to its recognition as a leading vector candidate for gene therapy applications. A number of Phase I and Phase II clinical trials utilizing AAV have been performed worldwide (Aucoin et al., Biotechnol. Adv. 26:73 (2008); Mueller et al., Gene Ther. 15:858 (2008)). However, many preclinical studies and successful clinical trials have demonstrated a number of challenges that will need to be addressed to sustain rAAV use for human gene therapy (Mueller et al., Gene Ther. 15:858 (2008)). One major challenge is establishing large scale manufacturing technologies in accordance with current Good Manufacturing Practices (cGMP) to yield the purified vector quantities needed for the expanding clinical need. The success of generating a scalable production technology relies heavily on understanding the basic biology of AAV in regard to generating reagents such as cell lines, plasmids or recombinant viral vectors, etc. that when used together, will closely mimic wild-type AAV production.
AAV has been classified as a dependovirus in the Parvovirus family because it requires coinfection with helper viruses such as adenovirus (Ad) or herpes simplex virus (HSV) for productive infection in cell culture (Atchison et al., Science 149:754 (1965); Buller et al., J. Virol. 40:241 (1981)). Parvoviruses are among the smallest of the DNA animal viruses with a virion of approximately 25 nm in diameter composed entirely of protein and DNA. The AAV genome is a linear, single-stranded DNA molecule containing 4679 bases (Srivastava et al., Virol. 45:555 (1983)). The wild-type (wt) AAV genome is made up of two genes that encode four replication proteins and three capsid proteins, respectively, and are flanked on either end by inverted terminal repeats (ITRs) (Lusby et al., J. Virol. 4:402 (1980); Srivastava et al., J. Virol. 45:555 (1983)). The ITRs are the only cis-acting elements necessary for genome replication and packaging into the capsid. The four replication proteins (Rep 78, 68, 52 and 40) are multifunctional and play a role in transcription, viral DNA replication and DNA packaging into the preformed viral capsid within the nucleus of the infected cell (Chejanovsky et al., Virology 173:120 (1989); King et al., EMBO J. 20:3282 (2001)). The viral capsid is made up of the three proteins Vp1, Vp2 and Vp3 in a ratio of 1:1:8 respectively. The capsid proteins are produced from the same open reading frame (ORF) but utilize different translational start sites.
Because the ITRs are the only cis acting elements necessary for genome replication and packaging, the rep and cap genes can be removed and cloned into a separate plasmid without a loss in function. A promoter and gene of interest driven by a promoter can then be cloned between the ITRs. Thus any gene that is flanked by the ITRs can effectively be packaged into an AAV capsid as long as the genome is smaller than 5.0 kb in size (Dong et al., Mol. Ther. 18:87 (2010); Grieger et al., J. Virol. 79:9933 (2005); Wu et al., Mol. Ther. 18:80 (2010)). However, AAV still lacks the ability to replicate. One of the distinctive features of AAV is the requirement of co-infection with a helper virus such as Ad or HSV. The generation of rAAV used to require transfection of the vector and packaging constructs into Ad-infected cells (Muzyczka, Curr. Top. Microbiol. Immunol. 158:97 (1992)). Upon co-infection with Ad or HSV, AAV utilizes several helper virus early genes to facilitate its own replication. Infection of Ad into producer cells to generate rAAV was effective in producing rAAV, but a consequence was that it also produced overabundant Ad particles. Complete removal of Ad has relied on physical techniques such as CsCl gradients, column chromatography, and a heat-denaturing step to inactivate any residual Ad particles that may still be present. While most of these procedures have succeeded to various degrees, the potential for Ad contamination is an unwanted risk and the presence of Ad denatured proteins is unacceptable for clinical use. A significant improvement in the evolution of rAAV production was the introduction of the triple plasmid transfection (Xiao et al., J. Virol. 72:2224 (1998)). This method used a variation of the rep and cap plasmid as well as the ITR plasmid, but eliminated the use of Ad infection. The Ad proteins of E1A, E1B, E4 and E2A and VA RNA were cloned into a single plasmid called XX680. Supplying the Ad helper genes on the XX680 plasmid eliminated Ad production in the transfected cells yielding only rAAV vector. The triple transfection method remains a standard production method in most laboratories experimenting with rAAV. However, this method has been limited to the use of adherent cells.
Advances in rAAV production have been made in the past 10 years that have allowed a number of laboratories to move away from production using adherent HEK293 cells and move toward scalable technologies such as infection-based technologies through the use of recombinant adenovirus (Gao et al., Mol. Ther. 5:644 (2002); Gao et al., Hum. Gene Ther. 9:2353 (1998); Liu et al., Mol. Ther. 2:394 (2000); Liu et al., Gene Ther. 6:293 (1999); Tessier et al., J. Virol. 75:375 (2001)), herpes simplex virus (Booth et al., Gene Ther. 11:829 (2004); Conway et al., Gene Ther. 6:986 (1999); Hwang et al., Mol. Ther. 7:S14 (2003); Kang et al., Gene Ther. 16:229 (2009); Thomas et al., Hum. Gene Ther. 20:861 (2009)), baculovirus expression vector system (BEVS) (Aslanidi et al., Proc. Natl. Acad. Sci. USA 106:5059 (2009); Cecchini et al., Gene Ther. 15:823 (2008); Kohlbrenner et al., Mol. Ther. 12:1217 (2005); Negrete et al., Meth. Mol. Biol. 433:79 (2008); Negrete et al., J. Gene Med. 9:938 (2007); Urabe et al., Hum. Gene Ther. 13:1935 (2002); Urabe et al., J. Virol. 80:1874 (2006)), and transient transfection of suspension HEK293 cells (Durocher et al., J. Virol. Meth. 144:32 (2007); Hildinger et al., Biotechnol. Lett. 29:1713 (2007); Park et al., Biotechnol. Bioeng. 94:416 (2006)). Park et al. and Durocher et al. demonstrated that approximately 1.4×104 and 3×104 vg/cell, respectively, were generated using their optimized serum-free suspension HEK293 cell production systems. Common with these studies is the fact that the yield of vector continues to be the impediment and is significantly below the vg/cell generated via transfection of adherent HEK293 cells and the rHSV production system.
Cesium chloride purification (CsCl) is still the most widely used form of AAV purification (Grieger et al., Nat. Protoc. 1:1412 (2006); Grieger et al., Adv. Biochem. Eng. Biotechnol. 99:119 (2005)). The benefits of using CsCl purification are its relative low cost, compatibility for any AAV serotype and separation of empty and genome containing particles. However, several drawbacks detract from this method being used for clinical applications including: (1) the time and effort required to identify the virus containing fractions within multiple CsCl gradient purification runs; (2) the resulting virus containing many impurities in addition to cesium; (3) impediments with scaling up; and (4) numerous open steps during purification (Brument et al., Mol. Ther. 6:678 (2002); Chahal et al., J. Virol. Meth. 139:61 (2007); Hermens et al., Hum. Gene Ther. 10:1885 (1999); Kaludov et al., Hum. Gene Ther. 13:1235 (2002); Smith et al., J. Virol. Meth. 114:115 (2003); Zolotukhin, Hum. Gene Ther. 16:551 (2005)). Experiments conducted by Hermens et al. and Zolotukin et al. showed that a density gradient medium called iodixanol was very effective in isolating AAV2 after further purification using heparin affinity chromatography (Hermens et al., Hum. Gene Ther. 10:1885 (1999); Zolotukhin et al., Gene Ther. 6:973 (1999); Zolotukhin et al., Methods 28:158 (2002)). The caveat of this system was the inability to purify other AAV serotypes and chimeric capsids lacking affinity for heparin sulfate, thus reverting purification of rAAV to CsCl. There have been several other attempts at purification without the use of CsCl including the manipulation of the viral capsids with epitopes (Koerber et al., Hum. Gene Ther. 18:367 (2007)) and various forms of chromatography (Brument et al., Mol. Ther. 6:678 (2002); Chahal et al., J. Virol. Meth. 139:61 (2007); Davidoff et al., J. Virol. Meth. 121:209 (2004); Gao et al., Hum. Gene Ther. 11:2079 (2000); Hermens et al., Hum. Gene Ther. 10:1885 (1999); Kaludov et al., Hum. Gene Ther. 13:1235 (2002); Smith et al., J. Virol. Meth. 114:115 (2003); Zolotukhin et al., Gene Ther. 6:973 (1999); Zolotukhin et al., Methods 28:158 (2002)). The use of ion exchange chromatography has been shown to successfully purify several AAV serotypes. Brument et al. and Davidoff et al. used a two column system to purify AAV serotypes 2, 5 and 8 (Brument et al., Mol. Ther. 6:678 (2002); Davidoff et al., J. Virol. Meth. 121:209 (2004)). Zolotukhin et al. showed that use of iodixanol in addition to ion exchange utilizing a Q-Sepharose column was able to purify serotypes 1, 2, and 5 (Zolotukhin et al., Methods 28:158 (2002)). These methods showed great promise in AAV purification, but were only effective on the serotypes specified. A universal method has yet to be identified that can purify all serotypes of AAV.
The present invention provides a HEK293 cell line that grows under animal component-free suspension conditions and can be used in an AAV production system that is rapid, scalable, produces high titers, and functions with all serotypes and chimeras of AAV.